Alzheimer’s disease

AD is the most common form of dementia. This non-communicable disorder begins in the hippocampal region of the brain and gradually spreads to other areas over time. Its progression manifests as impaired thinking ability, motor skills, learning, memory, and language functions, all of which arise from neuronal death and ultimately lead to severe dementia in multiple brain regions [9,10].

AD is characterized by two major pathological hallmarks: the formation of amyloid plaques and neurofibrillary tangles (NFTs), both of which contribute to neuronal degeneration. Amyloid plaques, also referred to as senile plaques, are formed by the amyloid-β (Aβ) peptide when it aggregates into dense fibrils outside neurons. This peptide is a byproduct of the amyloid precursor protein (APP), which plays an essential role in neuronal development. APP and the amyloid precursor-like proteins APLP1 and APLP2 in mammals, along with APPL in Drosophila, constitute a protein family whose members each span the membrane once and possess a large extracellular domain. Within this family, only APP can generate the amyloidogenic fragment [11-13].

Eight isoforms of APP have been identified, of which three are the most common: the 695-amino-acid, 751-amino-acid, and 770-amino-acid variants. The 695-amino acid isoform is predominantly expressed in the central nervous system, whereas the 751 and 770-amino acid isoforms are expressed more broadly across tissues. A major portion of the Aβ sequence is located within the extracellular N-terminal region of APP, while a smaller segment resides within its transmembrane domain. APP also contains an intracellular C-terminal region that includes a highly conserved motif composed of the amino acids Tyr, Thr, Pro, Asn, Glu, and Tyr, referred to as the “YENPTY” sequence [14,15].

APP undergoes proteolytic processing via two alternative and mutually exclusive pathways. In the non-amyloidogenic pathway, α-secretase cleaves APP within the Aβ region, releasing a soluble extracellular fragment (sAPPα) and generating an 83-amino-acid C-terminal fragment (C83). Subsequent cleavage of C83 by γ-secretase produces the non-pathogenic P3 peptide along with the APP intracellular domain (AICD). In contrast, the amyloidogenic pathway is initiated by β-secretase cleavage, yielding a soluble extracellular fragment (sAPPβ) and a 99-amino-acid C-terminal fragment (C99). γ-Secretase processing of C99 then results in the generation of Aβ peptides of varying lengths, together with the same intracellular fragment, AICD, thereby linking this pathway to AD pathology [14,16,17].

APP plays a crucial role in a wide range of physiological processes, including cell-cell adhesion and interactions, binding to the extracellular matrix and cytoskeleton, regulation of cell growth and survival, neuronal migration and motility, synapse formation, neurite outgrowth, synaptic plasticity, and the proper distribution of neurons within the brain [18]. In addition to these essential functions, APP undergoes proteolytic processing mediated by secretase enzymes, a process that can lead to the generation of Aβ peptides under amyloidogenic conditions [19]. Within the full-length APP molecule, the Aβ region adopts an α-helical conformation; however, following proteolytic cleavage and peptide release, this region undergoes a conformational transition into β-sheet-rich structures that favour aggregation [20]. Aβ peptides range from 36 to 43 amino acids in length, with Aβ40 and Aβ42 being the most abundant and biologically relevant species, the latter being particularly associated with increased aggregation propensity and neurotoxicity [21].

Aβ peptides are inherently hydrophobic, a property that strongly favours their tendency to self-associate through hydrophobic interactions, leading to progressive assembly into a spectrum of aggregated species. Initially, monomeric Aβ molecules misfold and oligomerize into low-molecular-weight oligomers, which can further associate into protofibrils and eventually into highly ordered fibrils that deposit extracellularly as amyloid plaques in AD brain tissue. This hierarchical aggregation process is driven by β-sheet-rich conformations stabilized by inter- and intramolecular interactions, with hydrophobic segments of the peptide playing a critical role in nucleation and growth of these assemblies. Importantly, soluble oligomers and protofibrillar assemblies, rather than the insoluble plaque cores themselves, are now widely recognized as the principal neurotoxic species, capable of interacting with neuronal membranes and receptors, disrupting synaptic function and plasticity, and initiating downstream pathways that contribute to synaptic degeneration and cognitive decline in AD [22-24].

A β-strand is a structural element of a polypeptide chain comprising approximately 3 to 10 amino acids arranged in an extended conformation. Multiple β-strands can associate laterally via inter-strand hydrogen bonds to form β-sheets, which are stabilized by regular hydrogen-bonding patterns between backbone amide and carbonyl groups. In the context of amyloidogenic proteins, the supramolecular assembly of β-sheets into stacked arrays is a fundamental step in the formation of amyloid fibrils and higher-order protein aggregates observed in amyloidoses, including AD. In these fibrils, monomeric Aβ peptides align perpendicular to the fibril axis, adopting a characteristic cross-β architecture in which β-strands from adjacent molecules run parallel to one another, and their lateral associations are reinforced by extensive hydrogen bonding networks. Although Aβ peptides may accumulate intracellularly under certain pathological conditions, their predominant localization in AD is extracellular, where they contribute to plaque deposits and disrupt normal tissue architecture [25,26].

The Aβ peptide can undergo several post-translational modifications (PTMs), including oxidation, racemization, isomerization, pyroglutamate formation at glutamate residues, and phosphorylation. Phosphorylated Aβ at Ser8 and pyroglutamate-modified Aβ exhibit similar aggregation behaviour and strongly promote the formation of oligomers and fibrillar Aβ assemblies. Such modifications increase the biochemical stability of Aβ and elevate its concentration within the brain [27].

Between monomeric Aβ peptides and mature fibrillar structures, a range of intermediate assemblies exists that either arise during fibril formation or represent structural transition states. In the Alzheimer’s brain, these intermediates include protofibrils and low‑ and high‑molecular-weight oligomers. Dimers participate in the fibril-forming pathway, whereas trimers and globulomers remain stable off‑pathway species that do not proceed toward fibril formation [28].

The role of soluble oligomeric species of Aβ in fibril formation was proposed more than two decades ago, and their toxic effects are well established today. In the Alzheimer’s brain, oligomers accumulate within astroglia and are subsequently taken up by microglia. Intraneuronal accumulation of Aβ is less frequently observed in elderly individuals with Down syndrome and in the late stages of AD, where amyloid plaques are predominantly found extracellularly. In contrast, during the early stages of the disease and in individuals with Down syndrome, intraneuronal accumulation of Aβ oligomers has been reported [29,30].

Extracellular aggregates of Aβ peptides assemble into senile plaques, which are a pathological hallmark observed throughout multiple regions of the AD brain. This aberrant deposition reflects an imbalance between Aβ production and elimination, rather than solely increased synthesis. In early AD, cerebrospinal fluid concentrations of Aβ-particularly Aβ42-are reduced, while insoluble Aβ42 accumulates within brain parenchyma, indicating impaired clearance mechanisms [31,32].

Several enzymatic and transporter-mediated systems contribute to extracellular Aβ removal. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are key Aβ-degrading proteases that metabolize soluble and aggregated Aβ peptides in the extracellular space, and reductions in their activity correlate with increased plaque burden in AD. Additionally, efflux transporters at the blood-brain barrier, including the ATP-binding cassette transporter P-glycoprotein (ABCB1) and the low-density lipoprotein receptor-related protein 1 (LRP1) facilitate transcytosis of Aβ from brain interstitial fluid into the circulation. In contrast, the receptor for advanced glycation end products (RAGE) mediates the influx of circulating Aβ into the brain. Alterations in the expression or function of these clearance components, namely decreased NEP/IDE activity, downregulation of P-glycoprotein and LRP1, and upregulation of RAGE impair Aβ efflux, promoting extracellular accumulation and plaque formation characteristic of AD pathology [33-35].

Abnormal, extracellular, fibrillar protein deposits found in tissues and organs are collectively referred to as amyloid. Amyloid is insoluble, adopts a β-sheet-rich fibrillar structure, and typically lacks structural, supportive, or motile functions, but it is associated with the pathology observed in a wide range of disorders collectively known as amyloidoses. These diseases, including AD, spongiform encephalopathies, and type II diabetes, are progressive conditions with high morbidity and mortality [36,37].

Aβ plays several physiological roles in the body, including enhancing synaptic plasticity, learning, and memory by promoting long-term potentiation in the hippocampus; modulating calcium dynamics; exerting antimicrobial and antiviral activity; facilitating recovery after brain injury; sealing blood-brain barrier leaks; and potentially suppressing cancer through effects on tumour growth and apoptosis. Additional proposed functions include regulation of cholesterol transport, kinase activation, transcriptional modulation, and participation in apoptosis. An imbalance between Aβ production and its clearance from brain tissue leads to extracellular accumulation of Aβ. Aβ also reduces ATP production and disrupts mitochondrial function, and this mitochondrial impairment contributes to the progression of AD [38,39].

NFTs are one of the principal neuropathological hallmarks of AD and play a critical role in neuronal dysfunction and death. These tangles consist of intracellular aggregates of abnormally hyperphosphorylated tau protein that assemble into paired helical filaments and straight filaments within neurons. Under normal physiological conditions, tau stabilizes microtubules and supports axonal transport; however, excessive phosphorylation reduces its affinity for microtubules, leading to cytoskeletal destabilization and impaired neuronal function. In AD brains, tau is approximately three to four times more hyperphosphorylated than in healthy brains and aggregates into the filamentous structures that form NFTs [40,41].

To date, more than 70 phosphorylation sites have been identified on tau, but the most critical residues involved in abnormal phosphorylation include Thr18, Ser199, Ser202, Thr205, Thr231, Ser396, and Ser422. The accumulation of hyperphosphorylated tau disrupts microtubule stability, impairs axonal transport, induces oxidative stress and mitochondrial dysfunction, and ultimately contributes to synaptic failure and neuronal degeneration. Among the various kinases implicated in tau dysregulation, glycogen synthase kinase-3 (GSK3), particularly its β isoform (GSK3β), is considered the principal enzyme mediating tau phosphorylation and hyperphosphorylation [42,43].